Gene Arrangements in Expression Vector Affect 3-Hydroxypropionic Acid Production in Klebsiella pneumoniae

Abstract

Biosynthesis of 3-hydroxypropionic acid (3-HP) typically involves two sequential reactions catalyzed by glycerol dehydratase (DhaB) and aldehyde dehydrogenase (AldH). Although plasmid-dependent over-expression of the two enzymes is common, systematic investigation of gene arrangement in vector has not been reported. Here we show that gene arrangements have a noticeable influence on 3-HP production. Using Klebsiella pneumoniae as a host, three AldH-coding genes: ald4 from Saccharomyces cerevisiae, aldh from Escherichia coli, and puuC from host K. pneumoniae, were respectively ligated to dhaB. The recombinant Kp/pET-pk-ald4-dhaB (Kp refers to as K. pneumoniae, pk is a native promoter) produced the highest yield of 3-HP in comparison to both Kp/pET-pkdhaB-ald4 and Kp/pET-pk-dhaB-pk-ald4, suggesting that the preferential expression of AldH can increase 3-HP production. Additionally, when different AldH-coding genes were respectively ligated downstream of dhaB, the recombinant Kp/pET-pk-dhaB-puuC produced more 3-HP than that by Kp/pET-pk-dhaB-aldh or Kp/pET-pk-dhaBald4, implying the intrinsic compatibility of native gene puuC with its host. These findings indicate the applicability of native AldH-coding gene and provide insights into strategies for metabolic engineering of multiple genes.

Production of 3-Hydroxypropanoic Acid From Glycerol by Metabolically Engineered Bacteria

“…The rationale here is that the gene adjacent to the promoter in an operon is normally more expressed. Specifically, the arrangement favoring aldehyde dehydrogenase expression led to a higher 3-HP production due to the diminished build-up of 3-HPA (Li et al, 2013a). Different promoters for driving the expression of aldehyde dehydrogenase gene has also been successfully evaluated (Li et al, 2016).…”

Biorefinery for Glycerol Rich Biodiesel Industry Waste

“…Fermentation of CG to 3-hydroxypropionic acid (3-HP) was obtained by recombinant Klebsiella pneumoniae carrying gene for aldehyde dehydrogenase of Saccharomyces cerevisiae [116,117]. Here, it was realized that preferential expression of ald4 from S. cerevisiae is the most instrumental gene regulating 3-HP yield.…”

“…Here, it was realized that preferential expression of ald4 from S. cerevisiae is the most instrumental gene regulating 3-HP yield. Further enhancements were achieved through ligation of puuc gene from K. pneumoniae [117]. Lactobacillus reuteri metabolizes glycerol through CoA-dependent propanediol utilization route leading to the production of 3-HP.…”

Biodiesel Industry Waste: A Potential Source of Bioenergy and Biopolymers

“…Fermentation of glycerol by Anaerobiospirillum has been shown to produce succinic acid [7], citric acid by Yarrowia lipolytica [8], 1,3-propanediol, acetic acid and butyric acid by Clostridium butyricum [9], and 3-hydroxypropionic acid by Klebsiella pneumoniae [10,11]. Whereas, Escherichia coli was able to metabolize it to a host of bio-products such as ethanol, succinate, acetate, lactate, high value omega-3 polyunsaturated fatty acids [12] and hydrogen (H 2 ) [13,14].…”